By contrast, the MMP inhibitor BB-3103 blocked the decrease in cell surface TNF-R in PMA-stimulated cells. with human peripheral blood monocytes. Other weak-base amines, including hydroxychloroquine, ammonium chloride and methylamine, also induced reduction of cell surface TNF receptors, whereas lysosomal proteinase inhibitor, leupeptin, and BB-3013 were without effect. Our results suggest that chloroquine down-regulates NS13001 cell surface TNF receptors by retarding their transport to the NS13001 cell surface, while cleavage of cell surface receptors is not inhibited by chloroquine. Introduction Tumour necrosis factor (TNF) is usually a pleiotropic cytokine that plays a critical role in immune and inflammatory responses.1 Overproduction of TNF has been implicated in a NS13001 number of pathological conditions, including septic shock, rheumatoid arthritis, Crohn’s disease, cerebral malaria, and multiple sclerosis, through the induction of other proinflammatory cytokines and cell adhesion molecules.2C6 TNF mediates its diverse effects through cell surface receptors. Two different types of TNF receptors (TNF-R), p55 and p75, have been identified as members of TNF-R superfamily.7,8 Rabbit Polyclonal to ELOA3 The p55 TNF-R is expressed ubiquitously on the surface of most cell types, while the p75 TNF-R is expressed primarily in haematopoietic cells and endothelial cells. 1 The extracellular regions of both TNF-R contain four common cysteine-rich domains and bind TNF with high affinity.7C9 The cytoplasmic regions of p55 and p75 TNF-R are quite distinct and transmit different but overlapping signals. Both receptors were reported to induce activation of nuclear factor (NF)-B and to be involved in TNF-mediated apoptosis.1,9 Gene knockout studies indicated that p55 TNF-R plays an essential role in mediating the TNF signals in lethal endotoxaemia and in non-specific immunity to infection,10 while p75 TNF-R suppresses TNF-mediated inflammatory responses.11 Diverse inflammatory stimuli such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS) and TNF itself, induce the shedding TNF-R from the surface, generating soluble TNF-R.12C14 The proteases responsible for the cleavage of TNF-R have not yet been identified, but matrix metalloproteinase (MMP) inhibitors blocked the shedding of both p55 and p75 TNF-R, and resulted in retention of these molecules around the cell surface.15,16 The TNF–converting enzyme (TACE), which cleaves membrane-bound pro-TNF to release mature soluble form, has been implicated in the cell surface shedding of p75 TNF-R.17 Antimalarial drugs such as chloroquine and hydroxychloroquine are known to have anti-inflammatory effects, and have long been used in the treatment of rheumatoid arthritis and lupus erythematosus.18C20 Some of the effects of chloroquine in these diseases seem to appear through inhibition of proinflammatory cytokine production, since chloroquine was shown to block TNF and interleukin-6 (IL-6) synthesis in stimulated human monocytes and mouse macrophages.21C23 In our previous study, chloroquine was shown to inhibit LPS-induced TNF synthesis in mouse macrophage RAW 264.7 cells, mainly by blocking conversion of cell-associated pro-TNF to a soluble mature form, rather than by inhibiting induction of TNF mRNA or production of pro-TNF.24 Here we examined the effect of chloroquine around the synthesis and metabolism of TNF-R in human histiocytic U-937 cells. In PMA-stimulated cells, chloroquine reduced the level of soluble and cell surface TNF-R, while cell-associated TNF-R was increased by chloroquine. Chloroquine had no effect on the level of p55 and p75 TNF-R mRNA. Other lysosome-inhibitory weak-base amines also reduced cell surface expression of TNF-R. These results suggest that chloroquine down-regulates cell surface TNF-R level by interfering with intracellular trafficking of TNF-R mediated by a pH-sensitive cellular process. Materials and methods Reagents and antibodiesChloroquine (diphosphate salt), PMA, ammonium chloride, methylamine, leupeptin, monensin and brefeldin A were purchased from Sigma Chemical Co. (St Louis, MO). BB-3103 was kindly provided by British Biotech Pharmaceuticals (Oxford, UK) and hydroxychloroquine sulphate (IntaPort Company Inc., Ridgewood, NJ) was a gift from Yuhan Industrial Co. (Seoul, South Korea). Mouse monoclonal and goat polyclonal antibodies against human p55 and p75 TNF-R, recombinant human soluble p55 and p75 TNF-R were purchased from R & D Systems (Minneapolis, MN). Cell cultureU-937, a human.